Activation-induced cytidine deaminase (AID) initiates two processes of programmed mutagenesis undertaken by activated B cells that vastly increase the efficacy of antibody responses, whether in the context of infection or vaccination. In somatic hypermutation (SHM), AID diversifies the antigen-binding repertoire of antibodies by creating initiating lesions that are converted into point mutations within the immunoglobulin loci variable region exons, the exons that encode the antigen-binding domain. In class switch recombination (CSR), AID activity in immunoglobulin heavy chain switch regions leads to DNA double strand breaks (DSBs) that provide intermediates for exchange of expressed IgH constant region exons, which determine pathogen- elimination effector function. We lack a definitive understanding of two functionally critical aspects of AID activity: how it is targeted to specific DNA regions, and how AID activity leads to different outcomes during SHM and CSR. This is a fundamental gap in our understanding of antibody maturation processes. Recent findings that the variable regions of broadly neutralizing antibodies against HIV (anti-HIV bnAbs) are extensively and necessarily hypermutated underscore the relevance of understanding these mechanisms. We now propose to test the hypothesis that the DNA sequence of target substrates promotes specific AID targeting and influences the outcome of such targeting, and, in this context, to elucidate in detai the roles of particular sequences and motifs of the immunoglobulin heavy chain variable regions exons (Aim 1) and switch regions (Aim 2). To this end, we developed a novel V(D)J passenger allele mouse model system and mutation mapping tools that efficiently measure AID mutation activity within a given DNA sequence. We will use hypotheses-driven approaches in which candidate sequence motifs are embedded in synthetic sequences and tested in our passenger allele system for effects on AID activity. In parallel, bioinformatics analyses of mutation and DSB data will be performed to discover novel motifs. These studies should provide a wealth of basic insights into the mechanism of SHM and CSR and thus into the mechanisms that generate diverse and potent antibodies. Information will also be relevant to our understanding of off-target AID activity that contributes to oncogenic mutations and chromosomal translocations. Beyond that, proposed experiments may contribute to designing approaches to optimize immune responses such as those that promote or direct the maturation of highly mutated anti-HIV bnAbs along desired pathways.